Stimulation of Bovine Whole-Blood Samples Cultured in Media Supplemented with Recombinant Interleukin-7 (IL-7) and IL-12 Extends the Life Span of the Gamma Interferon Assay To Detect Mycobacterium bovis-Infected Cattle

Abstract

ABSTRACT The gamma interferon (IFN-γ) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples from Mycobacterium bovis -infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative from M. bovis , and incubated at 37°C in 5% CO 2 in air. Plasma was removed and assayed for an IFN-γ response using bovine IFN-γ enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-γ responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-γ assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication.